Our studies have focused on the the development of vaccines and the testing of antivirals. Using our pathogenic molecular clones (SIV/Mne) obtained from a pigtailed macaque naturally infected with AIDS, we have also examined the virologic and molecular changes in SIV that are associated with progression to AIDS. The time interval between infection and death, expressed as a precentage of total life span, is very similar between macques infected with these SIV clones and humans infected with HIV-1. These SIV/Mne viruses have therefore been used by several investigators as challenge stocks for a variety of AIDS vaccine protocols. 1. Role of cellular antigens in AIDS vaccines. Macaques immunized with uninfected human cells have been shown to be protected from challenge with SIV propagated in human cells. In collaboration with Drs. L. Arthur and L. Henderson, we have shown that various cellular antigens (including beta-2 microglobulin (beta-2M), HLA class I and HLA class II DR) are specifically associated with purified preparations of HIV and SIV. To identify the potential antigens involved in protection, macaques were immunized with beta-2M or with HLA class I or class II DR purified from H9 cells. Although all macaques developed humoral immune responses to these proteins, only the macaques immunized with HLA DR were protected from a subsequent challenge with HIV grown in H9 cells. This was the first demonstration that immunization with a cellular protein can protect from an AIDS virus infection. The study described above represents xenoimmunization - the use of human HLA molecules to immunize macaques. These studies have now been extended to alloimmunization studies - a study of the role of macaque class II antigens in protecting from AIDS virus infection (alloimmunization studies). In collaboration with Dr. M. Carrington, we have identified class II alleles in the pigtailed macaque colony at the University of Washington, and obtained class II DR from microvesicles harvested from Herpesvirus papio transformed macaque B-lymphocytes. Macaques that share similar and distinct class II alleles (as determined by oligotyping) were immunized with microvesicles containing DR, and then challenged with SIV/Mne grown in lymphocytes from the animal from which the class II antigen was purified. Animals have been monitored for evidence of viral infection, plasma loads of viral RNA and for the development of anibodies to SIV. All macaques became infected - antibodies to class II did not correlate with protection from infection in an alloimmunization regimen. The reasons why protection was evident in the xenoimmunization but not in the alloimmunization experiment are being pursued. Possibilities include the different species of macaques used in the two studies, and the manner in which the cellular antigens were presented. 2. Recombinant SIV vaccines. We previously reported that immunization with recombinant SIVmne envelope (gp160) vaccines protected macaques against intravenous challenge by the cloned homologous virus, E11S, but protection was only partially effective (43%) against an uncloned stock of SIVmne. To examine the role of core antigens in inducing or broadening protective immunity against intravenous challenges by heterologous viruses, we immunized macaques with either envelope, core proteins, or both antigens formulated as a mixture. Animals immunized with core antigens alone were not protected even against the homologous virus challenge, although the virus load was 100-fold lower than that of control (non-immunized) animals, suggesting an important role for core antigen-specific immune responses in controlling the acute infection. Animals that received both envelope and core antigens were now protected against an intravenous heterologous virus challenge, indicating that responses to core antigens contributed to the broadening of protective immunity. The results from these systematic studies argue strongly for the inclusion of multiple antigens in the design of recombinant vaccines against AIDS. 3. Role of cell-mediated immunity in AIDS vaccines. A possible role for cell-mediated immunity (CMI) in protection against HIV infection is suggested by the finding that a significant number of potentially HIV-exposed individuals from different risk groups, who show no evidence of infection, exhibit a strong HIV-specific CMI. To test this hypothesis in the SIV-macaque model, we inoculated macaques with graded doses of SIV and examined these animals for SIV viral sequences and the development of immune responses. Macaques inoculated with doses of SIV below the threshold required for virus isolation or seroconversion exhibited lymphocyte proliferation and were protected from an infectious SIV challenge delivered 16 months later. Additional studies are planned that will compare both aldrithiol-2 inactivated and live virus delivered at a similar subinfectious dose. 4. Development of primate vaccines that protect against natural type D retorvirus infection. Infectious type D retroviruses (SRVs) produce a naturally occurring and often fatal disease originally observed among macaques housed at primate centers in the United States. Five neutralization types have been identified (SRV1 - SRV5), three of which have been molecularly cloned and sequenced (SRV-1, 2, and 3). These infectious type D retroviruses cause an often-fatal immunosuppressive disease in macaques that is manifested by progressive weight loss, persistent diarrhea, anemia, thymic and lymphoid atrophy, opportunistic infections, and unusual neoplasms. Since the experimental infection of macaques with SIV results in similar clinical outcomes, animals used in AIDS experiments must be free of these SRV retroviruses so as not to confound pathogenesis and vaccine studies. It has therefore become important to eradicate and prevent type D retrovirus infections among captive, group-housed macaques maintained at primate centers. The primary objective of these experiments is to develop a safe and efficacious vaccine against SRV that can be used by primate centers in order to protect these valuable animals from these naturally occurring infections. We have previously reported that a vaccinia virus, recombined with and expressing the envelope proteins of our molecular clone of SRV-2, completely protected immunized macaques from a challenge with up to one million infectious SRV-2 particles delivered intravenously. This report was the first to describe that a recombinant vaccine could completely protect against a primate retrovirus infection. Future studies are now planned to test whether this vaccine will protect in a more natural setting; for example, among group-housed macaques in a colony setting. Macaques will be immunized with recombinant vaccinia virus expressing the envelope glycoproteins of SRV-2, SRV-1, and both recombinant vaccines and subsequently exposed to animals naturally infected with SRV-2. Control macaques, similarly exposed, will be immunized with wild-type vaccinia virus. Animals that are protected from this natural challenge will then be exposed to macaques infected with other type D retroviruses (SRV-1), in an effort to determine the limits and the correlates of the protective immunity elicited by these vaccines. Future experiments may also include recombinant vaccinia virus expressing Gag and Pol viral proteins to measure whether the inclusion of these additional antigens broadens the protective response as they do for SIV vaccines.